The present invention relates to a treatment of pulmonary fibrosis which can be caused by a variety of agents and may be associated with a number of diseases. The most common form of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). IPF is a progressive and lethal pulmonary disease occurring in between about 9 to 23 people per 100,000 and for which no cure is presently available. Although most physicians generally treat IPF and other fibrotic lung diseases with high doses of corticosteroids, a favourable response rarely occurs, and if it does occur, it is transient. Instead, the use of high dose corticosteroids leads to a variety of complications that can be lethal themselves.
It has been demonstrated that a protein called transforming growth factor-beta (TGF-.beta.) is important in causing the inflammation and progressive scar tissue in pulmonary fibrosis. The presence of inflammation and scarring lead to morbidity and mortality. TGF-.beta. exists in three isoforms in mammals, designated as TGF-.beta..sub.1, TGF-.beta..sub.2 and TGF-.beta..sub.3. The characteristics as well as the in vitro and in vivo biological effects are most extensively reported for TGF-.beta..sub.1. Applicants have found that the TGF-.beta..sub.1 isoform is important in the pathogenesis of pulmonary fibrosis induced by bleomycin. All cells in the body produce TGF-.beta.s and all cells can respond to it. However, it is usually secreted non-covalently bound to a latency associated peptide (LAP) which renders it biologically inactive. The inactive form is called latent TGF-.beta..sub.1 (L-TGF-.beta..sub.1).
Applicants have found that in an animal model of pulmonary fibrosis and in samples from patients with IPF, TGF-.beta..sub.1 is secreted by alveolar macrophages in an active form. As well, applicants have found indications that in advanced cases of IPF, active TGF-.beta..sub.1 is secreted by epithelial cells. Epithelial cells may be the source of TGF-.beta..sub.1 found subepithelially where there is extensive fibrous connective tissue present. The subepithelial location of TGF-.beta..sub.1 could result in expansion of the fibroblast cell population and enhanced connective tissue synthesis and therefore play a critical role in the pathogenesis of pulmonary fibrosis.
Others have shown that, even in the presence of corticosteroids, once active TGF-.beta..sub.1 is present, it causes fibrosis to occur. Corticosteroids are able to prevent macrophages from entering the lungs, however, applicants have shown that once macrophages are able to secrete active TGF-.beta..sub.1, the presence of huge doses of corticosteroids does not inhibit the process. Furthermore, epithelial cells are structural cells and their number is not affected by corticosteroids. Secretion of TGF-.beta..sub.1 by epithelial cells is unaffected by corticosteroids, and this fact may explain the relative failure of corticosteroids in treating IPF.
The present invention provides means to prevent macrophages (and other cells) from secreting the active form of TGF-.beta..sub.1, thus stopping the inflammation and fibrosis from progressing. Therefore, the present invention may also be useful in the treatment of other diseases that have been associated with abnormal production of TGF-.beta..sub.1. These include but are not limited to scleroderma, systemic lupus erythematosis, sarcoidosis, silicosis, asbestosis, tuberculosis, hypersensitivity pneumonitis, keloids, burn induced hypertrophic scarring, cirrhosis of the liver, hypertrophic vetrinopathy, rheumatoid arthritis, Chron's disease, metastatic breast cancer, and other malignancies.
It is of significant clinical importance that:
(1) Although systemic corticosteroids which are standard therapy for the above mentioned diseases decrease the influx of inflammatory cells, they do not alter the secretion of active TGF-.beta..sub.1 by macrophages.
(2) In the presence of high systemic corticosteroids, if active TGF-.beta..sub.1 is present, it reverses all possible inhibition of enhanced connective tissue synthesis induced by steroids.
(3) The use of high systemic corticosteroids results in severe side effects, morbidity, and possibly even death. The applicants findings suggest that:
I) TGF-.beta..sub.2 and TGF-.beta..sub.3 proteins (that are the other forms of TGF-.beta.) are ubiquitously expressed, and PA1 ii) TGF-.beta..sub.3 may be important in physiological responses to injury, while PA1 iii) TGF-.beta..sub.1 protein expression is associated with normal and aberrant tissue repair.
These observations would suggest that in disorders mediated by excessive inflammation dominated by or regulated by macrophages (examples of which disorders are given above) and aberrant expression of TGF-.beta..sub.1, there would be a favourable outcome if the effects of TGF-.beta..sub.1 were inhibited. An approach to doing this may be by preventing latent-TGF-.beta..sub.1 (L-TGF-.beta..sub.1) from being activated.
Macrophages are mononuclear phagocytes derived from the bone marrow. Although macrophages are recognized for their ability to phagocytose foreign particles and tissue debris, macrophages also have an important role in wound repair. At sites of injury, prior to connective tissue synthesis, there is an influx of activated macrophages. When activated, macrophages secrete a number of pro-inflammatory and fibrogenic cytokines such as platelet derived growth factor (PDGF), interleukin-1 (IL-1), IL-6, tumour necrosis factor-alpha (TNF-.alpha.), basic fibroblast growth factor (bFGF), as well as TGF-.beta.. Of these cytokines, TGF-.beta. is one of the most potent regulators of inflammation and connective tissue synthesis.
In the context of wound repair, TGF-.beta..sub.1 is a potent chemoattractant for macrophages and induces these cells to express PDGF, IL-1, b-FGF, TNF-.alpha. and TGF-.beta..sub.1 itself. These effects of TGF-.beta..sub.1 on macrophages suggest that TGF-.beta..sub.1 at a site of injury can result in a macrophage dominated inflammatory infiltrate and subsequently enhanced connective tissue synthesis.
The main cellular source of most connective tissue proteins are fibroblasts. In vitro, TGF-.beta..sub.1 induces proliferation of immature fibroblasts and is a potent chemoattractant of mature fibroblasts and thereby in vivo could increase the number of fibroblasts in an area where TGF-.beta..sub.1 is present. Furthermore, TGF-.beta..sub.1 induces fibroblasts and other cells to synthesize, secrete and stabilize extracellular matrix proteins such as the collagens.
TGF-.beta..sub.1 is synthesized as a large 390 amino acid precursor that undergoes a number of intracellular processing steps that include cleavage of the LAP from the mature TGF-.beta..sub.1 protein. However, with rare exception when TGF-.beta..sub.1 is secreted by cells, it remains non-covalently associated in a 1:1 ratio with its LAP. The non-covalent association of TGF-.beta..sub.1 with its LAP renders the TGF-.beta..sub.1 biologically inactive. Since TGF-.beta..sub.1 and its receptors are ubiquitously expressed and since TGF-.beta..sub.1 has numerous biological effects, the ability of a cell to activate L-TGF-.beta..sub.1 upon secretion may be an important regulatory mechanism of TGF-.beta..sub.1 action in vivo. In vitro, the LAP can be dissociated from the mature peptide by a number of non-physiological conditions such as extremes of pH, boiling, and chaotropic agents. Other substances that may be more physiological, such as plasmin and thrombospondin-1, can also activate L-TGF-.beta..sub.1. In addition, a variety of substances, when cultured in the presence of certain cell lines, can induce the cultured cells to secrete active TGF-.beta.. For example, when high levels are present of D-glucose in cultures of a murine mesangial cell line or glucocorticoids in cultures of an osteoblast-like cell line, there is secretion of active TGF-.beta. into the conditioned media (CM). The physiological relevance of this post-translational activation of L-TGF-.beta. in these in vitro systems is unclear.
In a well characterized rat model of pulmonary injury and fibrosis,. induced by the antineoplastic antibiotic, bleomycin, it has been demonstrated that total lung TGF-.beta. was markedly increased seven days after bleomycin administration, when it was localized almost exclusively to alveolar macrophages. When alveolar macrophages were prevented from entering the lungs with high systemic doses of corticosteroids, this enhanced expression of total lung TGF-.beta. was abrogated. It has also been demonstrated that soon after bleomycin administration, explanted alveolar macrophages were induced to generate a biologically active form of TGF-.beta..sub.1 and plasmin, the generation of which was maximal seven days after bleomycin administration.
Furthermore, the secretion of active TGF-.beta..sub.1 was totally inhibited by the presence of alpha.sub.2 -antiplasmin, a naturally occurring inhibitor of plasmin. When large quantities of plasmin were added to activated alveolar macrophages, there was further activation of the L-TGF-.beta..sub.1. However, when plasmin was added to the L-TGF-.beta..sub.1 present in cell-free conditioned media from the same alveolar macrophages, no further activation of L-TGF-.beta..sub.1 occurred. Other findings suggest that the generation of plasmin is important in the post-translational activation of alveolar macrophage derived L-TGF-.beta..sub.1 during an inflammatory pulmonary injury response and that the activation required the presence of intact macrophages. Alveolar macrophages isolated from human patients with IPF also demonstrated inhibited production of active TGF-.beta..sub.1 in the presence of alpha.sub.2 -antiplasmin.
Applicants have now found that activation of L-TGF-.beta..sub.1 involves L-TGF-.beta..sub.1 /TSP-1 complex which interacts with the TSP-1 receptor, CD36, to process L-TGF-.beta..sub.1 to the mature form in the presence of plasmin. It has also been found that synthetic CD36 peptides can be used to prevent activation of TGF-.beta..sub.1 in rat and in human alveolar macrophages, thereby controlling the inflammation process. The present invention, in accordance with these findings, relates to activation of L-TGF-.beta..sub.1 and regulation thereof.
The present invention will be more readily illustrated with reference to the following description.